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The GP Ib complex can participate in endothelial cell (EC) migration on von Willebrand factor (vWF) or the mixed matrix of vWF and type I collagen (vWF/collagen). In this study, viper venom proteins alboaggregin (albo) A or B blocked GP Ibalpha, and echistatin inhibited alphavbeta3 binding. Albo A, B and echistatin inhibited EC migration on vWF and vWF/collagen. Albo B or the anti-GP Ibalpha monoclonal antibody (mAb) 1b1 did not affect the migration of smooth muscle cells or fibroblasts, which lack GP Ib. EC also migrate on albo A- or albo B-coated dishes. PD98059, which blocks ERK activation, abolished EC migration on vWF, vWF/collagen, collagen or albo B. Soluble albo A or 1b1 dramatically inhibited ERK activation during EC migration on vWF or albo B. Echistatin inhibited ERK activation on vWF and vitronectin (VN), but not albo B. Thus, in addition to alphavbeta3, EC GP Ibalpha initiates ERK activation, and regulates ERK-induced EC migration on vWF.

We have previously shown that, in addition to the vitronectin receptor (VNR, alpha(v)beta(3)), the GP Ib complex can participate in endothelial cell (EC) attachment to von Willebrand Factor (vWF) (D. A. Beacham, M. S. Cruz, and R. I. Handin, 1995, Thromb. Haemostas. 73, 309-317; D. A. Beacham, L.-P. Tran, and S. S. Shapiro, 1997, Blood 89, 4071-4077). In this study we have investigated the functional roles of these vWF receptors in the migration of untreated and TNFalpha-treated EC on vWF, a mixture of vWF and type I collagen, and on vitronectin (VN). In agreement with previous studies (D. I. Leavesley, M. A. Schwartz, M. Rosenfeld, and D. A. Cheresh, 1993, J. Cell Biol. 121, 163-170), the migration of untreated and TNFalpha-treated EC on VN was dependent entirely on the VNR. Migration of untreated EC on vWF was inhibited 10-15% by recombinant vWF-A1, the GP Ibalpha-binding domain on vWF which abrogates the platelet GP Ibalpha-vWF interaction. In contrast, migration of TNFalpha-treated EC on vWF was inhibited 50-60% by vWF-A1 or the anti-GP Ibalpha mAb AS-7 but only 20% by the anti-VNR mAb LM609. On a mixed vWF-collagen substratum, vWF-A1 inhibited untreated EC migration by 45%, and TNFalpha-treated EC migration by 75%. The possible role of EC proliferation was eliminated, since hydroxyurea completely inhibited EC proliferation without reducing migration significantly. The anti-GP Ibalpha mAb Ib1 inhibited EC migration by 50%, but reduced proliferation by only 15%. Taken together, our data demonstrate that EC migration on vWF-containing substrata involves the GP Ib complex as well as the VNR and raises the possibility that the VNR and GP Ib act cooperatively in supporting EC migration., (Copyright 1999 Academic Press.)

Exposure to shear stress has been shown to alter the expression of a number of surface components of cultured endothelial cells (EC). However, relatively few studies have examined the status of human EC surface proteins after prolonged flow, more closely corresponding to the steady state in vivo. Since the promoter region of glycoprotein (Gp) Ib alpha contains several copies of a putative shear stress response element, 5'-GAGACC-3', we investigated the response of cultured human umbilical vein EC (HUVEC) GpIb alpha to shear stress over a 72 h time period. In response to 30 dynes/cm2 of shear stress, total cell content of GpIb alpha protein was markedly increased above static levels at 7 and 24 h, as determined immunohistochemically. Western blot analysis of whole cell lysates after 24, 48, and 72 h of shear treatment demonstrated a 2.4-, 4.1-, and 3.2-fold increase in total GpIb alpha protein, respectively. Cell surface protein expression of GpIb alpha increased 2.5-fold at 7 h, as measured by quantitative immunofluorescence, and remained at that level at 24 h. After 48 h of shear stress, cell surface GpIb alpha, GpIX, and GpV, analyzed by flow cytometric analysis, were further increased over the levels observed at 24 h. The increase in cell surface membrane expression of GPIb alpha at 24, 48, and 72 h was confirmed by immunoprecipitation of biotinylated surface proteins. No upregulation of GpIb alpha was noted after exposure to shear stress of 1-3 dynes/cm2. These observations imply that under steady-state arterial shear conditions endothelial expression of the GpIb complex is significantly greater than observed in static EC cultures, and raise the possibility of a more important role for this complex under flow, rather than static conditions.

Endothelial cells (EC) possess at least two membrane receptors for von Willebrand factor (vWF), the vitronectin receptor (VNR, alpha(v)beta3), which recognizes an Arg-Gly-Asp (RGD) sequence in the C-terminus of vWF, and glycoprotein Ib alpha (GP Ib alpha), which interacts with a region in the N-terminal A1 domain of vWF. In the absence of added cytokines, EC attachment to a vWF substratum is mediated largely through the alpha(v)beta3, with a smaller contribution by GP Ib alpha. In the present study, we have examined the effect of cytokines on the receptor specificity of EC attachment to wild-type vWF (WT-vWF) and to vWF, which had been mutated in the C-terminal RGDS sequence (RADS-vWF). Exposure of human umbilical vein EC (HUVEC) to tumor necrosis factor-alpha (TNF-alpha) or to TNF-alpha in combination with interferon-gamma (IFN-gamma), but not to interleukin-1beta (IL-1), increased attachment to RADS-vWF by about twofold. The TNF-alpha-induced increase in EC attachment was accompanied by an increase in cell surface GP Ib alpha expression; GP Ib alpha surface expression was not increased by IL-1. Attachment of untreated HUVEC to WT-vWF could be inhibited 60% to 70% by a monoclonal antibody (MoAb) (LM609) to the VNR and 30% to 40% by the A1 fragment of vWF (containing the GP Ib alpha binding domain). The pattern of inhibition of attachment to WT-vWF was largely unchanged after TNF-alpha treatment of HUVEC. In contrast, the attachment to WT-vWF of HUVEC, treated with TNF-alpha +IFN-gamma was completely inhibited by vWF-A1 and inhibited only 35% by the anti-VNR antibody LM609. Two MoAbs to GP Ib alpha produced similar, but incomplete, inhibition. Pretreatment of HUVEC with the combination of TNF-alpha + IFN-gamma produced a dramatic decrease in VNR expression, confirming previous findings of Defilippi et al. These results suggest that in the presence of the inflammatory cytokines TNF-alpha + IFN-gamma, the endothelial GP Ib complex is a major determinant of HUVEC adhesion to surface-bound vWF.